Novel pyrazinone and pyridinone thrombin inhibitors incorporating weakly basic heterobicyclic P1-arginine mimetics

Article abstract of DOI:10.1016/j.ejmech.2005.03.007

The design, synthesis and biological activity of new thrombin inhibitors with a pyridinone or pyrazinone core and different heterobicyclic P₁ arginine side-chain mimetics are described. The arginine side-chain mimetics used in this study are (±

Full text of DOI:10.1016/j.ejmech.2005.03.007

European Journal of Medicinal Chemistry 40 (2005) 782–791
www.elsevier.com/locate/ejmech
Original article
Novel pyrazinone and pyridinone thrombin inhibitors incorporating
weakly basic heterobicyclic P1-arginine mimetics
Andreja Kranjc a, Lucija Peterlin Mašicˇ a, Sebastjan Reven a, Klemen Mikic a, Andrej Preželj b,
Mojca Stegnar c, Danijel Kikelj a,*
a Faculty of Pharmacy, University of Ljubljana, Aškercˇeva 7, 1000 Ljubljana, Slovenia
b Lek Pharmaceuticals, Verovškova 57, 1526 Ljubljana, Slovenia
c University Medical Centre, Department of Angiology, Zaloška 7, 1525 Ljubljana, Slovenia
Received 3 December 2004; revised and accepted 7 March 2005
Available online 10 May 2005
Abstract
The design, synthesis and biological activity of new thrombin inhibitors with a pyridinone or pyrazinone core and different heterobicyclic
P1 arginine side-chain mimetics are described. The arginine side-chain mimetics used in this study are ( )-4,5,6,7-tetrahydro-2H-indazol-5-
ylmethanamine and both enantiomers thereof, ( )-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine and the corresponding R enantiomer.
Compound 25, the most potent in the series of pyrazinone inhibitors, exhibited a Ki of 41 nM in vitro and high selectivity against trypsin and
factor Xa.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Thrombin inhibitors; Arginine side chain mimetics; Enantiomers; Stereoselective action
1. Introduction
tion, antithrombin agents are an important component in the
treatment of patients with acute coronary syndromes and
venous thromboembolism [3]. The main limitation of the
clinically used indirect thrombin inhibitors heparin and low
molecular weight heparins, as well as of the natural occuring
thrombin inhibitor hirudin (Ki = 22 fM), its recombinant ana-
logs lepirudin and desirudin, synthetic hirulog bivalirudin and
the non-peptide low molecular weight inhibitor argatroban,
is their requirement for parenteral application. Consequently
they are used for acute and not for chronic therapy. Enor-
mous efforts have been dedicated over the last decades
towards the design of potent and specific small molecule
thrombin inhibitors having a wide safety margin and appro-
priate physicochemical and pharmacokinetic properties per-
mitting oral dosing [4–12]. Ximelagatran, a very recently
approved prodrug of the thrombin inhibitor melagatran, ful-
fils, to a certain extent, the listed goals and could replace war-
farin, a vitamin K antagonist used for almost 50 years for
chronic treatment of some thrombotic indications [13].
While the prodrug approach has been successfully em-
ployed to produce P1 benzamidine-based thrombin inhibitors
with acceptable oral bioavailability, the majority of recent
approaches in the development of oral thrombin inhibitors
The pathophysiological role of an excessively active coagu-
lation cascade, with subsequent inappropriate thrombus for-
mation in blood vessels is well recognized. Venous thrombo-
sis, which often leads to pulmonary embolism, is frequently
triggered by vascular injury at the time of surgery or trauma
[1]. In contrast, arterial thrombosis may lead to acute coro-
nary syndromes, often as a result of plaque rupture. The cur-
rent paradigm is that thrombosis is the major reason for com-
plications of atherosclerosis, such as myocardial infarction
and stroke, and the major factor responsible for
atherosclerosis-related mortality [2]. Due to the central role
played by thrombin in the pathogenesis of thromboembolic
disorders, by triggering fibrin formation and platelet activa-
Abbreviations: DMF, N-N-dimethylformamide; EDC, N-ethyl-N’-(3-
dimethylaminopropyl)-carbodiimide; HOBT, 1-hydroxybenzotriazole.
* Corresponding author : Prof. Dr. Danijel Kikelj, University of Lju-
bljana, Faculty of Pharmacy, Aškercˇeva 7, 1000 Ljubljana, Slovenia; Tel.:
+386 1 4769561; fax: +386 1 4258031.
E-mail address: danijel.kikelj@ffa.uni-lj.si (D. Kikelj).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.03.007

A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
783
Fig. 1
.
Optimization strategy of 3-amino-6-methyl-2-pyridinone acetamide thrombin inhibitors.
have focused on compounds that incorporate less basic P1
moieties [14–18]. Thus, we have focused on the design and
synthesis of thrombin inhibitors that incorporate weakly basic,
partially saturated heterobicyclic P1-arginine side chain
mimetics [19–23] and have reported on a series of proline-
based thrombin inhibitors and 3-amino-2-pyridinone aceta-
mide thrombin inhibitors incorporating different lipophilic
residues in the P3 part of the molecule [24–29]. The pyridi-
none template was previously shown by other groups, in the
design of human leukocyte elastase inhibitors, thrombin
inhibitors, and tissue factor/factor VIIa inhibitors, to be an
appropriate peptidomimetic template that mimics the hydro-
gen bond array of the backbone of peptide inhibitors and pro-
vides a good fit of the inhibitor in the enzyme active site
[15,30–33]. As a replacement for the pyridinone core, the
pyrazinone scaffold has been frequently used [9,32,34] and it
has been observed by Merck scientists that this modification
improves the pharmacokinetic properties of thrombin inhibi-
tors. Further improvement of the pharmacokinetics of pyrazi-
none thrombin inhibitors was observed also on substitution
of the 6-methyl group by chlorine [9,32].
Fig. 2
.
Reagents and conditions: (a) L-(+)-tartaric acid, H20/EtOH (2:1),
75 °C, then 5 °C, 3 days; (b) D-(-)-tartaric acid, H20/EtOH (2:1), 75 °C, then
5 °C, 3 days; (c) 85% aq KOH, 10 °C.
Figs. 3,4 outline the elaboration of P3-P2 pyridinone and
P3-P2 pyrazinone synthons 6 [32] and 10 [9] and the ultimate
coupling reactions with P1-arginine mimetics which led to
target inhibitors 15-25. The coupling reactions were per-
formed using N-ethyl-N’-(3-dimethylaminopropyl)carbo-
diimide (EDC) and 1-hydroxybenzotriazole (HOBT), both in
dry N,N-dimethylformamide, as condensing reagents.
3. Pharmacology
In this article we report the optimization of our previously
described 3-amino-6-methyl-2-pyridinone acetamide throm-
bin inhibitors [27,28]. The optimization strategy included the
synthesis of single enantiomers and replacement of the pyri-
dinone core by the pyrazinone scaffold (Fig. 1). The arginine
side-chain mimetics of low basicity used in this study
comprise racemic 4,5,6,7-tetrahydro-2H-indazol-5-ylmetha-
namine (1), its two enantiomers 2 and 3, and 4,5,6,7-
tetrahydro-1,3-benzothiazole-2,6-diamine in racemic form (4)
and in the form of the (+)-enantiomer 5 (Fig. 1).
The ability of the new thrombin inhibitors to inhibit the
enzymatic activities of thrombin, trypsin and factor Xa was
measured as described previously [25] by amidolytic enzyme
assay using chromogenic substrates and is expressed as inhi-
bition constants, Ki [37].Values for Ki were calculated accord-
ing to Cheng and Prusoff [38] based on IC50 values, or from
a relation between reaction velocity equations in the absence
and presence of inhibitor, using the relevant Km [39]. The
2. Chemistry
We have recently reported a convenient synthetic approach
to the conformationally restricted arginine side chain mimet-
ics 1 and 4 [19–22,24]. In the synthesis of the enantiomers
(-)-4,5,6,7-tetrahydro-2H-indazol-5-ylmethanamine (2) and
(+)-4,5,6,7-tetrahydro-2H-indazol-5-ylmethanamine (3), opti-
cal resolution of ( )-1 with L- and D-tartaric acid, respec-
tively, was employed (Fig. 2). The resolution of ( )-2,6-
diamino-4,5,6,7-tetrahydrobenzothiazole (4) has already been
described [35].
Fig. 3. Reagents and conditions: (a) p-F-C6H4-CH2SO2Cl [36], CH2Cl2, Et3N,
0 °C to rt, 2 h; (b) HClg, EtOAc, 0 °C, 20 min; (c) arginine mimetic 1, 2 or 3,
EDC, HOBt, N-methyl-morpholine, DMF, rt, 12 h.

784
A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
Fig. 4
.
Reagents and conditions : (a) RCH2CH2NH2, Et3N, toluene/EtOH (3:1), 120 °C, 15 h; (b) N-chlorosuccinimide, 1,2-dichloroethane, reflux, 2,5 h; (c)
1 M aq KOH, dioxane, 3 h; (d) arginine mimetic 1, 2, 3, 4 or 5, EDC, HOBt, N-methylmorpholine, DMF, rt, 12 h.
selectivity for thrombin over trypsin was expressed as the ratio
i(trypsin)/Ki(thrombin)
in the series of pyridinone inhibitors incorporating 4,5,6,7-
K
.
tetrahydro-1,3-benzothiazol-2-amine as P1 arginine mimetic,
where the R enantiomer binds with 16-fold higher affinity
than the S enantiomer [28]. Comparison of these two series
of thrombin inhibitors leads to the conclusion that the abso-
lute configuration at C6 of a P1 partially saturated heterobi-
cyclic ring plays a less important role in the 4,5,6,7-tetrahydro-
2H-indazol-6-ylmethanamine series than in the 4,5,6,7-
tetrahydro-1,3-benzothiazole-2,6-diamine series.
4. Results and discussion
The in vitro inhibitory potencies of inhibitors 15-25 are
listed in Tables 1,2. Based on the crystal structure of throm-
bin inhibitor 15 complexed to human thrombin [27], which
indicated preferential binding of the R enantiomer to the active
site, we prepared the single enantiomers of its p-fluoro ana-
logue 16, which showed improved inhibitory potencies com-
pared to 15. The (+) enantiomer 18 (Ki = 47 nM) binded with
almost 2-fold higher affinity to the thrombin active site than
the (-) enantiomer 17 (Ki = 76 nM). Although the (-) enanti-
omer also showed good affinity for thrombin, that for the (+)
enantiomer was higher and showed 1815-fold selectivity
against trypsin and 1772-fold selectivity against factor Xa.
Preferential binding of the R enantiomer was also observed
In the pyrazinone series of thrombin inhibitors, we synthe-
sized analogues with 2-phenylethyl-amino-, 2-(4-
chlorophenyl)ethylamino- and 2-(2-pyridyl)ethylamino resi-
dues in the P3 part and two different P1 arginine side chain
mimetics 4,5,6,7-tetrahydro-2H-indazol-5-ylmethanamine
and 4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine. In this
series, the 4,5,6,7-tetrahydro-2H-indazol-5-ylmethanamine
and 4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine argin-
ine side chain mimetics in the P1 part of molecule contribute
Table 1
Inhibitory potencies of compounds 15-18
Compound
R1
R2
Ki (µM)
Trypsin
>500
Selectivity
Thrombin/Trypsin
Thrombin
FXa
15
0.17
103
>2941
16
17
0.057
0.076
113.2
87.9
58.2
1985
133.9
85.3
1761
18
0.047
83.3
1815

A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
785
Table 2
Inhibitory potencies of compounds 19-25
Compound
R1
R2
Ki (µM)
Trypsin
Selectivity
Thrombin/Trypsin
Thrombin
FXa
19
0,493
15,4
46,5
>100
138,5
31
20
21
0,745
1,33
>100
>100
62
>75
22
23
0,060
0,053
>100
>100
59,0
>1665
>1887
>100
24
25
0,085
0,041
>100
>100
121,5
82,5
>1176
>2440
Microanalyses;
18 Calcd.: C, 54.64; H, 5.58; N, 13.85
Found: C, 54.78; H, 5.79; N, 13.90;
19 HRMS (EI): Calcd.: 492.09125 Found: 492.09020;
20 Calcd.: C, 53.69; H, 5.19; N, 17.89
21 Calcd.: C, 58.73; H, 5.82; N, 18.68
22 Calcd.: C, 52.23; H, 4.82; N, 21.32
23 Calcd.: C, 52.23; H, 4.82; N, 21.32
24 Calcd.: C, 57.08; H, 5.47; N, 22.19
25 Calcd.: C, 54.84; H, 5.70; N, 21.32
Found: C, 54.08; H, 5.18; N, 17.56;
Found: C, 59.07; H, 5.94; N, 18.44;
Found: C, 52.25; H, 5.05; N, 20.99;
Found: C, 52.22; H, 5.05; N, 20.99;
Found: C, 56.84; H, 5.65; N, 21.84;
Found: C, 55.16; H, 5.75; N, 20.98.
similarly to the overall binding affinity of the inhibitors (com-
pounds 20, 21 and 22, 24 respectively)
Since the pyridinone inhibitor 15 (Ki = 170 nM; Table 1), with
a benzylsulfonyl group in the P3 part, exhibits an 8-fold higher
affinity for thrombin than the pyrazinone analogue 21
(Ki = 1.33 µM) incorporating the identical P1 moiety, it can
be concluded that the central heterocyclic P2 core and the
sulfonyl group have only weak influence on binding of the
inhibitors to the thrombin active site.
To determine the influence of chirality in the pyrazinone
series of thrombin inhibitors, we prepared single enanti-
omers 23 and 25 of racemic inhibitors 22 and 24 that both
incorporate a P3-pyridine moiety. The inhibitory potencies of
compound 22 and its R-enantiomer 23, possessing a 4,5,6,7-
tetrahydro-1,3-benzothiazole-2,6-diamine arginine mimetic in
the P1 part, are practically the same (22: Ki = 60 nM; 23:
Ki = 53 nM), suggesting that, in contrast to the similar pyri-
dinone series of inhibitors [28], the binding affinities of the
pyrazinone series are not strongly dependent on the absolute
Modifications in the P3 part of the pyrazinone series of
thrombin inhibitors demonstrated that, irrespective of the P1
moiety, replacement of the P3 phenyl ring with a 2-pyridyl
ring improved the potency of inhibitors (cf. 20 (Ki = 745 nM)
and 22 (Ki = 60 nM); 21 (Ki = 1.33 µM) and 24 (Ki = 85 nM);
From the crystal structure of a related thrombin inhibitor in a
complex with human thrombin [9], it is evident that an
electron-deficient P3 pyridine reinforces the edge-to-face d-p
interactions in the S3 pocket between the P3 pyridine group
and the p-rich Trp 215, providing a greater overall binding
affinity versus its phenyl counterpart. Introduction of the
p-chloro substituent (19; Ki = 493 nM) led to a slightly greater
binding affinity over the phenyl derivative 20 (Ki = 745 nM),
but the Ki value of 19 was still 8-fold lower than that of inhibi-
tor 22 (Ki = 60 nM) which incorporates pyridine as a P3 group.

786
A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
configuration at C6 of the heterobicyclic ring.A similar mini-
mal influence of chirality was also observed in the pyrazi-
none inhibitor 24 with a P3 2-(2-pyridyl)ethylamino moiety
and 4,5,6,7-tetrahydro-2H-indazol-5-ylmethanamine in the P1
part of the molecule. Thus, the (-) enantiomer 25 (Ki = 41 nM)
showed a 2-fold better binding affinity than the correspond-
ing racemic inhibitor 24 (Ki = 85 nM). Comparison of throm-
bin inhibitory potency and selectivity of our most potent com-
pound 25 (Ki = 41 nM; > 2400-fold selectivity versus trypsin)
and melagatran (Ki = 2 nM; 2-fold selectivity versus trypsin)
[40], an active form of a recently approved prodrug ximela-
gatran [41], shows that while possessing excellent selectivity
the thrombin inhibitory constant of compound 25 is of one
order of magnitude lower than that of melagatran. This is prob-
ably due to the presence of a bicyclic ring which hinders 25
from achieving optimal interaction with the enzyme active
site. On the other side, the bicyclic ring which enters the S1
pocket could be responsible for the observed high selectivity
versus trypsin. In the pyridazinone series the selectivity of
the inhibitors versus trypsin was generally high in com-
pounds with P3 2-(2-pyridyl)ethylamino moiety. All throm-
bin inhibitors listed in Tables 1,2 exhibited good (58- to 138-
fold) selectivity for inhibition of thrombin versus inhibition
of factor Xa. Thus, regarding inhibition of thrombin and fac-
tor Xa, they could be regarded as pure anti-thrombin type of
coagulation factors’ inhibitors.
THF was kept over sodium and distilled immediately prior to
use.Analytical TLC was performed on Merck silica gel (60 F
254) plates (0.25 mm), with visualization with ultraviolet light
and ninhydrin. Column chromatography was carried out on
Florisil® (particle size 100-200 mesh) and silica gel 60 (par-
ticle size 240-400 mesh). Melting points were determined on
a Reichert hot stage microscope and are uncorrected. 1H NMR
spectra were recorded on a Bruker AVANCE DPX300 spec-
trometer in CDCl3 or DMSO solution with TMS as the inter-
nal standard. IR spectra were obtained on a Perkin-Elmer
1600 FT-IR spectrometer. Microanalyses were performed on
a Perkin-Elmer C, H, N analyzer 240 C. Elemental analyses
were within 0.4% of the theoretical values. Mass spectra
were obtained using aVG-AnalyticalAutospec Q mass spec-
trometer. HPLC analyses were performed on Agilent Tech-
nologies HP 1100 instrument with G1365B UV-VIS detec-
tor, using a Eurospher C18 column (4.6 x 250 mm). The eluant
was a mixture of acetonitrile (60%) and 0.1 M ammonium
acetate buffer pH 4.15 (40%). Chemical names were gener-
ated using ACD/Name software.
6.1.2. Resolution of ( )-4,5,6,7-tetrahydro-2H-indazol-5-
ylmethanamine dihydrochloride (1×2HCl).
To an aqueous solution of the dihydrochloride of 1 (1.24 g,
5.53 mmol), 5 M solution of NaOH (1.12 ml) was added. The
resulting solution was extracted with dichloromethane
(4×40 ml), the organic layers collected and dried (Na2SO4)
and the solvent removed in vacuo, giving 0.65 g (78%) of 1
as a white solid. D-(-)-tartaric acid (0.65 g, 4.30 mmol) was
added to a suspension of 1 (0.65 g, 4.30 mmol) in 5 ml of
mixture of water/ethanol (2:1) and the mixture was heated to
reflux for 10 minutes. After cooling, the mixture was stood
for 3 days in a refrigerator. A precipitate formed and was col-
lected and recrystallized two times from a mixture of
water/ethanol (2:1). The crystalline D-(-)-tartrate was sus-
pended in 20 ml of water and concentrated aqueous HCl was
added dropwise until a clear solution resulted. After addition
of 85% aqueous KOH (0.57 ml) at 10 °C, the free base was
extracted with dichloromethane, the organic layers collected
and dried (Na2SO4), and the solvent removed in vacuo to yield
120 mg (18%) of (-)-4,5,6,7-tetrahydro-2H-indazol-5-
ylmethan-amine 2 as a white solid; mp 129-132 °C;
5. Conclusion
Optimization of our previously described 3-amino-6-
methyl-2-pyridinone acetamide thrombin inhibitors resulted
in potent and selective inhibitors 18 and 25. This study
revealed that the (+) enantiomer 18 of P1 4,5,6,7-tetrahydro-
2H-indazol-5-ylmethanamine compound 16, with Ki of 47 nM
and 1815-fold selectivity versus trypsin, binds to the throm-
bin active site with only 2-fold higher affinity than the (-)
enantiomer 17. In the pyrazinone series of inhibitors possess-
ing tetrahydroindazole in the P1 part, better binding affinity
was achieved with (-) enantiomer 25 than with the racemic
inhibitor 24. Compound 25 is a potent thrombin inhibitor,
with an in vitro Ki of 41 nM and over 2440-fold selectivity
against trypsin. As in the pyrazinone series the affinity for
thrombin improved markedly on replacement of the P3 ben-
zlysulfonyl group by the 2-(2-pyridyl)ethyl moiety, it can be
concluded that the 2-(2-pyridyl)ethyl group contributes impor-
tantly to the inhibitory potency of compounds 22, 23, 24 and
25.
␣
͓ ͔
20 = -16.69° (c = 0.29, H2O); 1H-NMR (300 MHz,
D
CDCl3): d = 1.36-1.50 (m, 1H, H-5), 1.87-2.10 (m, 2H, 2H-6),
2.12-2.23 and 2.62-2.72 (2×m, 4H, 2H-4, 2H-7), 2.80-2.87
(m, 2H, CH2NH2), 7.23 (s, 1H, H-3).
(+)-4,5,6,7-Tetrahydro-2H-indazol-5-ylmethanamine (3)
was obtained from 1 (0.38 g, 1.70 mmol) using L-(+) tartaric
acid (0.26 g, 1.70 mmol) and following the same procedure
as described above; yield 70 mg (28%),
␣
20 = +15.36°
͓ ͔
6. Experimental protocols
(c = 0.25, H2O); The product was in al other resDpects identi-
cal to 2.
6.1. Chemistry
6.1.1. Materials and methods
Chemicals were obtained from Aldrich Chemical Co.,
Fluka and Synthetech and used without further purification.
6.1.2.1. tert-Butyl 2-[3-{[(4-fluorobenzyl)sulfonyl]amino}-6-
methyl-2-oxo-1(2H)-pyridinyl]acetate (7). (4-Fluorophenyl)
methanesulfonyl chloride (1.59 g, 7.62 mmol) was added in

A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
787
portions to a stirred solution of tert-butyl (3-amino-6-methyl-
2-oxo-1(2H)-pyridinyl)acetate (6) [32] (1.30 g, 5.45 mmol)
and triethylamine (1.5 ml, 10.78 mmol) in dichloromethane
(15 ml) cooled to 0 °C.After two hours 10% KHSO4 solution
(15 ml) was added and the aqueous phase was extracted twice
with dichloromethane. The pooled organic phases were dried
over Na2SO4, filtered and the solvent evaporated under
reduced pressure. The product was purified by column chro-
matography (silica gel, CH2Cl2/MeOH = 40:1) to give 750 mg
(34%) of a white solid; mp 149-151 °C; IR (KBr): m 3142,
2982, 1742, 1652, 1595, 1511, 1449, 1353, 1225, 1155, 896,
772, 577, 496 cm–1. MS (FAB): 411 (MH+, 73%), 154
(100%). 1H-NMR (300 MHz, CDCl3): d = 1.54 (s, 9H, t-Bu),
2.29 (s, 3H, 6-CH3), 4.30 (s, 2H, p-F-C6H4- CH2), 4.76 (s,
2H, NCH2), 6.04 (d, 1H, J = 7.54 Hz, H-5), 6.98-7.05 (m,
2H, 2×CH), 7.16-7.21 (s br, 1H, NHSO2), 7.23-7.29 (m, 2H,
2×CH) 7.38 (d, 1H, J = 7.54 Hz, H-4).
1443, 1355, 1223, 1157, 771, 571 cm–1. MS (FAB): 488
1
(MH+, 11%), 73 (100%). H-NMR (300 MHz, CDCl3):
d = 1.50-1.59 (m, 1H, CH-5), 1.90-2.23 (m, 2H, 6-CH2), 2.18-
2.26, 2.62-2.69, 2.71-2.78 (3×m, 4H, 4-CH2, 7-CH2), 2.48
(s, 3H, CH3), 3.23-3.38 (m, 2H, CH2NH), 4.29 (s, 2H, p-F-
C6H4CH2), 4.65 (s, 2H, NCH2), 6.09 (d, 1H, J = 7.54 Hz,
Py-H-5), 6.80-6.89 (m, 2H, CH, 2-NH), 6.91-6.99 (m, 2H,
2×CH), 7.21-7.29 (m, 3H, 3-CH, NHCO, CH), 7.38 (d, 1H,
J = 7.54 Hz, Py-H-4) 8.05 (s, 1H, NHSO2); HPLC: purity
96.5%; Anal. C23H26FN5O4S × H2O (C, H, N).
6.1.2.4. (-)-2-[3-{[(4-Fluorobenzyl)sulfonyl]amino}-6-methyl-
2-oxo-1(2H)-pyridinyl]-N-(4,5,6,7-tetrahydro-2H-indazol-5-
ylmethyl)acetamide (17). Using the general procedure
described above, 17 was prepared from 2-[3-{[(4-fluoro-
benzyl)-sulfonyl]amino}-6-methyl-2-oxo-1(2H)-pyridinyl]-
acetic acid (8) (117 mg, 0.33 mmol) and (-)-4,5,6,7-
tetrahydro-2H-indazol-5-ylmethanamine (2) (50 mg,
0.33 mmol); yield 40 mg (25%), white powder; HPLC: purity
6.1.2.2. 2-[3-{[(4-Fluorobenzyl)sulfonyl]amino}-6-methyl-2-
oxo-1(2H)-pyridinyl]acetic acid (8). HCl gas was bubbled
through a stirred suspension of tert-butyl 2-[3-{[(4-fluoro-
benzyl)-sulfonyl]amino}-6-methyl-2-oxo-1(2H)-pyridinyl]-
acetate (7) (745 mg, 1.82 mmol) in ethylacetate cooled to
0 °C, until a clear solution was obtained. The HCl-saturated
clear solution was stirred for one hour at room temperature
and evaporated to give a brown solid; yield: 606 mg (94%),
mp 117-120 °C; IR (KBr): m 3142, 1714, 1654, 1601, 1510,
1458, 1370, 1227, 1152, 1032, 879, 584 cm–1. MS (FAB):
355 (MH+, 72%), 154 (100%). 1H-NMR (300 MHz, DMSO-
d6): d = 2.27 (s, 3H, CH3), 4.53 (s, 2H, p-F-C6H4- CH2), 4.78
(s, 2H, NCH2), 6.11 (d, 1H, J = 7.54 Hz, H-5), 7.11-7.20 (m,
3H, 2×CH, H-4), 7.37-7.44 (m, 2H, 2×CH), 8.66 (s, 1H,
NHSO2), 13.13 (s, 1H, COOH).
89.3%;
␣
20 = -10.00° (c = 0.11, CH3OH); The product
͓ ͔
was in all othDer respects (mp, IR, MS, NMR) identical to com-
pound 18;
6.1.2.5. ( )-2-[3-{[(4-Fluorobenzyl)sulfonyl]amino}-6-
methyl-2-oxo-1(2H)-pyridinyl]-N-(4,5,6,7-tetrahydro-2H-
indazol-5-ylmethyl)acetamide (16). Using the general
procedure described above, 16 was prepared from 2-[3-{[(4-
fluorobenzyl)-sulfonyl]amino}-6-methyl-2-oxo-1(2H)-pyri-
dinyl]acetic acid (8) (106 mg, 0.30 mmol) and ( )-4,5,6,7-
tetrahydro-2H-indazol-5-ylmethanamine dihydrochloride
(1.HCl) (67 mg, 0.30 mmol); yield 40 mg (27%), white pow-
der; HPLC: purity 89.8%; The product was in all respects
(mp, IR, MS, NMR) identical to compound 18.
6.1.2.3. (+)-2-[3-{[(4-Fluorobenzyl)sulfonyl]amino}-6-
methyl-2-oxo-1(2H)-pyridinyl]-N-(4,5,6,7-tetrahydro-2H-
indazol-5-ylmethyl)acetamide (18). General procedure for
the synthesis of compounds 16-18 by coupling of P3-P2
fragment 8 with ( )-4,5,6,7-tetrahydro-2H-indazol-5-
ylmethanamine (1), (-)-4,5,6,7-tetrahydro-2H-indazol-5-
ylmethanamine (2) and (+)-4,5,6,7-tetrahydro-2H-indazol-
5-ylmethanamine (3). 2-[3-{[(4-Fluorobenzyl)sulfonyl]-
amino}-6-methyl-2-oxo-1(2H)-pyridinyl]acetic acid (8)
(160 mg, 0.45 mmol) and (+)-4,5,6,7-tetrahydro-2H-indazol-
5-ylmethanamine (3) (68 mg, 0.45 mmol) were dissolved in
N,N-dimethylformamide (1.0 ml). HOBT (61 mg, 0.45 mmol)
and, after adjusting the pH to 8 with N-methylmorpholine,
EDC (86 mg, 0.45 mmol) were added. The reaction mixture
was stirred overnight at room temperature. It was then parti-
tioned between ethyl acetate and saturated aqueous NaHCO3,
and the organic layer washed with brine, dried over Na2SO4
and filtered and the solvent removed under vacuum. The crude
product was purified by column chromatography (silica gel,
CH2Cl2/MeOH = 9:1) to give 57 mg (26%) of a faintly brown
6.1.2.6. Ethyl 2-[3-[(4-chlorophenethyl)amino]-2-oxo-1(2H)-
pyrazinyl]acetate (11a). General procedure for the synthe-
sis of compounds 11a-c by reaction of ethyl 2-[3-bromo-2-
oxo-1(2H)-pyrazinyl]acetate (10) with 2-(4-chlorophenyl)-
1-ethanamine, 2-phenyl-1-ethanamine and 2-(2-pyridinyl)-
1-ethanamine. A solution of 2-(4-chlorophenyl)-1-ethanamine
(417 mg, 2.68 mmol), triethylamine (0.37 ml, 2.68 mmol)
and ethyl 2-[3-bromo-2-oxo-1(2H)-pyrazinyl]acetate (10)
(700 mg, 2.68 mmol) in a mixture of 2 ml toluene and 0.35 ml
ethanol was heated under reflux overnight. The mixture was
concentrated and the residue partitioned between dichlo-
romethane (11 ml) and saturated aqueous NaHCO3 (11 ml).
The aqueous layer was washed with dichloromethane
(4x10 ml) and the combined organic layers dried over Na2SO4.
The solvent was removed at reduced pressure to give a yel-
low solid that was purified by column chromatography (silica
gel, CH2Cl2/MeOH = 20:1) to give 372 mg (41%) of 11a as a
white solid which was used without further purification for
the synthesis of 12a. IR (KBr): m 3322, 2989, 1742, 1649,
1608, 1563, 1495, 1374, 1217, 1016, 757 cm–1. MS (FAB):
336 (MH+, 32%), 75 (100%). 1H-NMR (300 MHz, CDCl3):
d = 1.31 (t, 3H, J = 7.16 Hz, CH3), 2.93 (t, 2H, J = 7.16 Hz,
powder; mp 116-119 °C;
␣
20 = +12.50° (c = 0.095,
͓ ͔
CH3OH); IR (KBr): m 3295, 2924,D1652, 1601, 1569, 1508,

788
A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
p-ClC6H4CH2), 3.67 (dt, 2H, J1 = 7.16 Hz, J2 = 6.02 Hz,
CH2NH), 4.27 (q, 2H, J = 7.16 Hz, CH2O), 4.56 (s, 2H,
CH2CO), 6.16 (s br, 1H, NH), 6.40 (d, 1H, J = 4.52 Hz, Pz-H),
6.89 (d, 1H, J = 4.52 Hz, Pz-H), 7.18 (d, 2H, J = 8.53 Hz,
2×CH), 7.29 (d, 2H, J = 8.53 Hz, 2×CH).
The combined organic layers were dried over Na2SO4, and
the solution concentrated under reduced pressure. The crude
product was purified by column chromatography (silica gel,
CH2Cl2/MeOH = 50:1) to give 178 mg (43%) of 12a as a
white solid which was used without further purification for
the synthesis of 13a; IR (KBr): m 3351, 2938, 1753, 1639,
1572, 1485, 1210, 1090, 1014, 808, 660 cm–1. MS (EI): 369
6.1.2.7. Ethyl 2-[2-oxo-3-(phenethylamino)-1(2H)-pyrazinyl]-
acetate (11b). Using the general procedure described above,
ethyl 2-[2-oxo-3-(phenethylamino)-1(2H)-pyrazinyl]acetate
(11b) was prepared from 2-phenyl-1-ethanamine (279 mg,
2.30 mmol), triethylamine (0,32 ml, 2,30 mmol) and ethyl
2-[3-bromo-2-oxo-1(2H)-pyrazinyl]acetate (15) (600 mg,
2,30 mmol). The crude product was purified by column chro-
matography (silica gel, CH2Cl2/MeOH = 9:1) to give 517 mg
(75%) of 11b as a pink solid which was used without further
purification for the synthesis of 12b. IR (KBr): m 3330, 2988,
1746, 1648, 1558, 1373, 1206, 1033, 703 cm–1. MS (EI): 301
1
(M+, 21%), 244 (100%). H-NMR (300 MHz, CDCl3):
d = 1.32 (t, 3H, J = 7.16 Hz, CH3), 2.92 (t, 2H, J = 7.16 Hz,
p-ClC6H4CH2), 3.66 (dt, 2H, J1 = 7.15 Hz, J2 = 6.03 Hz,
CH2NH), 4.28 (q, 2H, J = 7.16 Hz, CH2O), 4.90 (s, 2H,
CH2CO), 6.07 (t, 1H, J = 5.61 Hz, NH), 6.98 (s, 1H, Pz-H),
7.18 (d, 2H, J = 8.52 Hz, 2×CH), 7.29 (d, 2H, J = 8.52 Hz,
2×CH).
6.1.2.10. Ethyl 2-[6-chloro-2-oxo-3-(phenethylamino)-1(2H)-
pyrazinyl]acetate (12b). Using the general procedure
described above 12b was prepared from ethyl 2-[2-oxo-3-
(phenethylamino)-1(2H)-pyrazinyl]acetate (11b) (326 mg,
1.08 mmol) and N-chlorosuccin-imide (147 mg, 1.10 mmol);
yield 363 mg (100%), red solid. The product was used with-
out further purification in the next step for the synthesis of
13b. IR (KBr): m 3351, 2985, 1756, 1648, 1573, 1486, 1210,
1033, 700 cm–1. MS (EI): 335 (M+, 33%), 244 (100%).
1H-NMR (300 MHz, CDCl3): d = 1.31 (t, 3H, J = 7.16 Hz,
CH3), 2.95 (t, 2H, J = 7.17 Hz, Ph-CH2), 3.71 (dt, 2H,
J1 = 6.98 Hz, J2 = 6.0 Hz, CH2NH), 4.28 (q, 2H, J = 7.16 Hz,
CH2O), 4.90 (s, 2H, CH2CO), 6.10 (t, 1H, J = 6.0 Hz, NH),
6.98 (s, 1H, Pz-H), 7.21-7.36 (2×m, 5H, Ph-H).
1
(M+, 37%), 210 (100%). H-NMR (300 MHz, CDCl3):
d = 1.31 (t, 3H, J = 7.13 Hz, CH3), 2.96 (t, 2H, J = 7.12 Hz,
Ph-CH2), 3.70 (q, 2H, J = 6.96 Hz, CH2NH), 4.27 (q, 2H,
J = 7.13 Hz, CH2O), 4.56 (s, 2H, CH2CO), 6.19 (s br, 1H,
NH), 6.39 (d, 1H, J = 4.62 Hz, Pz-H), 6.90 (d, 1H,
J = 4.62 Hz, Pz-H), 7.22-7.36 (m, 5H, Ph-H).
6.1.2.8. Ethyl 2-[2-oxo-3-{[2-(2-pyridinyl)ethyl]amino}-
1(2H)-pyrazinyl]acetate (11c). Using the general procedure
described above, ethyl 2-[2-oxo-3-{[2-(2-pyridinyl)ethyl]-
amino}-1(2H)-pyrazinyl]acetate (11c) was prepared from
2-(2-pyridinyl)-1-ethanamine (715 mg, 5.85 mmol), triethy-
lamine (0.81 ml, 5.85 mmol) and ethyl 2-[3-bromo-2-oxo-
1(2H)-pyrazinyl]acetate (10) (1.53 g, 5.85 mmol); yield 1.56 g
(88%), brown solid. The crude product was used immedi-
ately without purification for the synthesis of 12c; IR (KBr):
m 3323, 2993, 2938, 1746, 1657, 1603, 1557, 1433, 1371,
1224, 1112, 990, 891, 780, 738 cm–1. MS (FAB): 303 (MH+,
6.1.2.11. Ethyl 2-[6-chloro-2-oxo-3-{[2-(2-pyridinyl)ethyl]-
amino}-1(2H)-pyrazinyl]acetate (12c). Using the general pro-
cedure described above, 12c was prepared from ethyl
2-[2-oxo-3-{[2-(2-pyridinyl)ethyl]amino}-1(2H)-pyrazinyl]-
acetate (11c) (1.08 g, 3.57 mmol) and N-chlorosuccinimide
(489 mg, 3.66 mmol); yield 1.19 g (98%), brown solid. The
product was used without further purification in the next step
for the synthesis of 13c. IR (KBr): m 3390, 3339, 2972, 2755,
1744, 1645, 1575, 1485, 1415, 1223, 1026, 777, 608 cm–1.
MS (FAB): 337 (MH+, 100%). 1H-NMR (300 MHz, CDCl3):
d = 1.30 (t, 3H, J = 7.16 Hz, CH3), 3.15 (t, 2H, J = 6.75 Hz,
Py-CH2), 3.85 (dt, 2H, J1 = 6.78 Hz, J2 = 6.40 Hz, CH2NH),
4.25 (q, 2H, J = 7.16 Hz, CH2O), 4.90 (s, 2H, CH2CO), 6.60
(t br, 1H, NH), 6.98 (s, 1H, Pz-H), 7.19 (m, 2H, 2×Py-H),
7.63 (m, 1H, Py-H), 8.60 (m, 1H, Py-H).
1
100%). H-NMR (300 MHz, CDCl3): d = 1.32 (t, 3H,
J = 7.16 Hz, CH3), 3.18 (t, 2H, J = 6.78 Hz, Py-CH2), 3.85
(dt, 2H, J1 = 6.78 Hz, J2 = 6.41 Hz, CH2NH), 4.25 (q, 2H,
J = 7.16 Hz, CH2O), 4.57 (s, 2H, CH2CO), 6.39 (d, 1H,
J = 4.71 Hz, Pz-H), 6.62 (t br, 1H, NH), 6.70 (d, 1H,
J = 4.71 Hz, Pz-H), 7.19 (m, 2H, 2×Py-H), 7.62 (m, 1H,
Py-H), 8.60 (d, 1H, J = 7.54 Hz, Py-H).
6.1.2.9. Ethyl 2-[6-chloro-3-[(4-chlorophenethyl)amino]-2-
oxo-1(2H)-pyrazinyl]acetate (12a). General procedure for
the synthesis of compounds 12a-c. N-chlorosuccinimide
(137 mg, 1.03 mmol) was added to a stirred solution of ethyl
2-[3-[(4-chlorophenethyl)amino]-2-oxo-1(2H)-pyrazinyl]-
acetate (11a) (372 mg, 1.11 mmol) in 1,2-dichloroethane
(6 ml) and the reaction mixture heated to reflux for 2.5 h.
During this time additional N-chlorosuccinimide was added
(13 mg, (0,097 mmol) after 1 h and 4 mg (0.030 mmol) after
1.5 h). The solution was then cooled to room temperature
and partitioned between dichloromethane (9 ml) and satu-
rated aqueous NaHCO3 (11 ml). The layers were separated
and the aqueous phase backwashed with CH2Cl2 (2 ×11 ml).
6.1.2.12. 2-[6-Chloro-3-[(4-chlorophenethyl)amino]-2-oxo-
1(2H)-pyrazinyl]acetic acid (13a). General procedure for
the synthesis of compounds 13a-13c. 1 M aqueous KOH
(0.96 ml, 0.96 mmol) was added to a stirred solution of ethyl
2-[3-[(4-chlorophenethyl)amino]-2-oxo-1(2H)-
pyrazinyl]acetate (12a) (178 mg, 0.48 mmol) in dioxane
(5 ml). After 3 h the solution was neutralized to pH 7 with
concentrated hydrochloric acid, and the mixture evaporated
under reduced pressure (toluene was added 2-3 times to form
azeotrope) to give 203 mg of a yellow solid containing 13a

A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
789
(164 mg, yield: 100%) and KCl. IR (KBr): m 3400, 1610,
1484, 1385, 1229, 1090, 804, 703 cm–1. MS (EI): 341 (M+,
7%), 216 (100%). 1H-NMR (300 MHz, DMSO-d6): d = 2.85
(t, 2H, J = 7.16 Hz, p-ClC6H4CH2), 3.49 (dt, 2H, J1 = 7.16 Hz,
J2 = 6.03 Hz, CH2NH), 4.29 (s, 2H, CH2CO), 6.84 (s, 1H,
Pz-H), 7.12 (t, 1H, J = 6.03 Hz, NH), 7.25 (d, 2H, J = 8.66 Hz,
2×CH), 7.34 (d, 2H, J = 8.66 Hz, 2×CH).
a white solid. The crude product was purified by column chro-
matography (silica gel, CH2Cl2/MeOH = 9:1) to give 37 mg
(yield: 35%) of 19 as a white powder; mp 234-237 °C. IR
(KBr): m 3346, 3274, 2936, 1644, 1586, 1530, 1490, 1231,
1091, 1014, 802 cm–1. MS (FAB): 493 (MH+, 24%), 154
(100%). 1H-NMR (300 MHz, DMSO-d6): d = 1.66-1.91 (m,
2H, CH2), 2.23-2.50 (m, 3H, 2×CH2), 2.72-2.82 (m, 1H, CH2),
2.86 (t, 2H, J = 7.16 Hz, p-ClC6H4-CH2), 3.51 (dt, 2H,
J1 = 7.16 Hz, J2 = 6.40 Hz, CH2NH), 3.96-4.08 (m, 1H,
CH-6), 4.70 (s, 2H, CH2CO), 6.65 (s, 2H, NH2), 6.94 (s, 1H,
Pz-H), 7.25 (d, 2H, J = 8.47 Hz, 2×CH), 7.32-7.41 (m, 1H,
Pz-NH), 7.34 (d, 2H, J = 8.47 Hz, 2×CH), 8.36 (d, 1H,
J = 7.53 Hz, NHCO). HRMS (EI) C21H22Cl2N6O2S: calcd.:
492.09125; found: 492.09020.
6.1.2.13. 2-[6-Chloro-2-oxo-3-(phenethylamino)-1(2H)-
pyrazinyl]acetic acid (13b). Using the general procedure
described above, 2-[6-chloro-2-oxo-3-(phenethylamino)-
1(2H)-pyrazinyl]acetic acid (13b) was prepared from ethyl
2-[6-chloro-2-oxo-3-(phenethylamino)-1(2H)-pyrazinyl]-
acetate (12b) (340 mg, 1.01 mmol) and 1 M aqueous KOH
(2.02 ml, 2.02 mmol). yield: 444 mg of a yellow solid con-
taining 13b (311 mg, yield: 100%) and KCl. IR (KBr): m 3366,
1617, 1574, 1483, 1373, 1314, 1234, 1110, 870, 699,
554 cm–1. MS (EI): 307 (M+, 29%), 216 (100%). 1H-NMR
(300 MHz, DMSO-d6): d = 2.86 (t, 2H, J = 7.07 Hz, PhCH2),
3.50 (dt, 2H, J1 = 7.07 Hz, J2 = 6.05 Hz, CH2NH), 4.29 (s,
2H, CH2CO), 6.84 (s, 1H, Pz-H), 7.08 (t, 1H, J = 6.05 Hz,
NH), 7.16-7.34 (m, 5H, Ph-H).
6.1.2.16. ( )-N-(2-Amino-4,5,6,7-tetrahydro-1,3-benzothia-
zol-6-yl)-2-[6-chloro-2-oxo-3-(phenethylamino)-1(2H)-
pyrazinyl]acetamide (20). Using the general procedure
described above, 20 was prepared from 2-[6-chloro-2-oxo-3-
(phenethylamino)-1(2H)-pyrazinyl]acetic acid (13b) (110 mg,
0.356 mmol) and ( )-4,5,6,7-tetrahydro-1,3-benzothiazole-
2,6-amine dihydrobromide (4×2HBr) (107 mg, 0.324 mmol).
The crude product was purified by column chromatography
(silica gel, CH2Cl2/MeOH = 9:1) to give 40 mg (27%) of
white solid; mp 242-246 °C. IR (KBr): m 3335, 3286, 2933,
1660, 1581, 1488, 1232, 695 cm–1. MS (FAB): 459 (MH+,
89%), 57 (100%). 1H-NMR (300 MHz, DMSO-d6): d = 1.69-
1.90 (m, 2H, CH2), 2.34-2.56 (m, 3H, 2×CH2), 2.71-2.81 (m,
1H, CH2), 2.87 (t, 2H, J = 7.53 Hz, Ph-CH2), 3.52 (dt, 2H,
J1 = 7.53 Hz, J2 = 6.40 Hz, CH2NH), 3.97-4.08 (m, 1H,
CH-6), 4.70 (s, 2H, CH2CO), 6.65 (s, 2H, NH2), 6.94 (s, 1H,
Pz-H), 7.17-7.37 (m, 6H, Ph, Pz-NH), 8.36 (d, 1H,
J = 7.91 Hz, NHCO) Anal. C21H23ClN6O2S×0,6 H2O (C, H,
N).
6.1.2.14. 2-[6-Chloro-2-oxo-3-{[2-(2-pyridinyl)ethyl]amino}-
1(2H)-pyrazinyl]acetic acid (13c). Using the general proce-
dure described above, 2-[6-chloro-2-oxo-3-{[2-(2-pyridinyl)-
ethyl]-amino}-1(2H)-pyrazinyl]acetic acid (13c) was prepared
from ethyl 2-[6-chloro-2-oxo-3-{[2-(2-pyridinyl)ethyl]-
amino}-1(2H)-pyrazinyl]acetate (12c) (1.16 g, 3.45 mmol)
and 1 M aqueous KOH (6.90 ml, 6.90 mmol). yield 1.01 g of
a yellow solid containing 13c (1065 mg, yield: 100%) and
KCl. IR (KBr): m 3339, 1709, 1654, 1581, 1484, 1379, 1392,
1114, 987, 772, 760 cm–1. MS (FAB): 309 (MH+, 100%).
1H-NMR (300 MHz, CDCl3): d = 3.02 (t, 2H, J = 7.53 Hz,
Py-CH2), 3.63 (dt, 2H, J1 = 7.53 Hz, J2 = 6.78 Hz, CH2NH),
4.38 (s, 2H, CH2CO), 6.85 (s, 1H, Pz-H), 7.21 (m, 2H, 2×Py-
H), 7.71 (m, 1H, Py-H), 8.50 (d, 1H, Py-H).
6.1.2.17. ( )-N-(2-Amino-4,5,6,7-tetrahydro-1,3-benzothia-
zol-6-yl)-2-[6-chloro-2-oxo-3-{[2-(2-pyridinyl)ethyl]amino}-
1(2H)-pyrazinyl]acetamide (22). Using the general procedure
described above, 22 was prepared from 2-[6-chloro-2-oxo-3-
{[2-(2-pyridinyl)ethyl]amino}-1(2H)-pyrazinyl]acetic acid
(13c) (95 mg, 0.308 mmol) and ( )-4,5,6,7-tetrahydro-1,3-
benzothiazole-2,6-amine dihydrobromide (4×2HBr) (93 mg,
0.280 mmol). The crude product was purified by column chro-
matography (silica gel, CH2Cl2/MeOH = 7:1) to give 54 mg
(42%) of tan solid; mp 246-249 °C. IR (KBr): m 3276, 2922,
1646, 1588, 1476, 1229, 1108, 747 cm–1. MS (FAB): 460
6.1.2.15. ( )-N-(2-Amino-4,5,6,7-tetrahydro-1,3-benzothia-
zol-6-yl)-2-[6-chloro-3-[(4-chlorophenethyl)amino]-2-oxo-
1(2H)-pyrazinyl]acetamide (19). General procedure for
the synthesis of compounds 19, 20, 22 and 23 by coupling
P3-P2 fragments 13 with ( )-4,5,6,7-tetrahydro-1,3-
benzothiazole-2,6-amine dihydrobromide (4×2HBr) and
(-)-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-amine (5). 2-[6-
Chloro-3-[(4-chlorophenethyl)amino]-2-oxo-1(2H)-pyra-
zinyl]acetic acid (13a) (81 mg, 0.236 mmol), ( )-4,5,6,7-
tetrahydro-1,3-benzothiazole-2,6-amine dihydrobromide
(4×2HBr) (71 mg, 0.215 mmol) and 1-hydroxybenzotriazole
(32 mg, 0.236 mmol) were dissolved in N,N-dimethyl-
formamide (1.0 ml), the pH adjusted to pH 8 with
N-methylmorpholine and 1-(3-dimethylaminopropyl)-3’-
ethyl-carbodiimide hydrochloride (45 mg, 0.236 mmol) then
added. The reaction mixture was stirred overnight at room
temperature, then diluted with saturated NaHCO3 (4.4 ml)
and water (6.6 ml) and the resulting precipitate filtered to yield
1
(MH+, 18%), 55 (100%). H-NMR (300 MHz, DMSO-d6):
d = 1.66-1.91 (m, 2H, CH2), 2.34-2.58 (m, 3H, 2×CH2), 2.71-
2.82 (m, 1H, CH2), 3.02 (t, 2H, J = 7.53 Hz, Py-CH2), 3.65
(dt, 2H, J1 = 7.53 Hz, J2 = 6.03 Hz, CH2NH), 3.95-4.04 (m,
1H, CH-6), 4.70 (s, 2H, CH2CO), 6.65 (s, 2H, NH2), 6.93 (s,
1H, Pz-H), 7.22 (ddd, 1H, J1 = 7.54 Hz, J2 = 4.90 Hz,
J3 = 1.13 Hz, Py-H-3), 7.27 (d, 1H, J = 7.54 Hz, Py-H-4),
7.41 (t, 1H, J = 6.03 Hz, Pz-NH), 7.71 (m, 1H, Py-H-5), 8.36
(d, 1H, J = 7.54 Hz, NHCO), 8.50 (m, 1H, Py-H-6). Anal.
C20H22ClN7O2S (C, H, N).

790
A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
6.1.2.18. N-[(6R)2-Amino-4,5,6,7-tetrahydro-1,3-benzothia-
zol-6-yl]-2-[6-chloro-2-oxo-3-{[2-(2-pyridinyl)ethyl]amino}-
1(2H)-pyrazinyl]acetamide (23). Using the general procedure
described above, 23 was prepared from 2-[6-chloro-2-oxo-3-
{[2-(2-pyridinyl)ethyl]amino}-1(2H)-pyrazinyl]acetic acid
(13c) (92 mg, 0.297 mmol) and (+)-4,5,6,7-tetrahydro-1,3-
benzothiazole-2,6-diamine (5) (46 mg, 0.270 mmol). The
crude product was purified by column chromatography (silica
gel, CH2Cl2/MeOH = 7:1) to give 30 mg (24%) of tan solid;
mp 207-212 °C. IR (KBr): m 3360, 2933, 1652, 1586, 1229,
1109, 746 cm–1. MS (FAB): 460 (MH+, 9%), 55 (100%).
1H-NMR (300 MHz, DMSO-d6): d = 1.66-1.91 (m, 2H, CH2),
2.34-2.58 (m, 3H, 2×CH2), 2.72-2.83 (m, 1H, CH2), 3.03 (t,
2H, J = 7.16 Hz, Py-CH2), 3.65 (dt, 2H, J1 = 7.16 Hz,
J2 = 6.03 Hz, CH2NH), 3.96-4.08 (m, 1H, CH-6), 4.70 (s,
2H, CH2CO), 6.65 (s, 2H, NH2), 6.94 (s, 1H, Pz-H), 7.22
(ddd, 1H, J1 = 7.54 Hz, J2 = 4.90 Hz, J3 = 1.13 Hz, Py-H-3),
7.27 (d, 1H, J = 7.54 Hz, Py-H-4), 7.41 (t, 1H, J = 6.03 Hz,
Pz-NH), 7.71 (m, 1H, Py-H-5), 8.36 (d, 1H, J = 7.53 Hz,
NHCO), 8.50 (m, 1H, Py-H-6).Anal. C20H22ClN7O2S (C, H,
N).
{[2-(2-pyridinyl)ethyl]amino}-1(2H)-pyrazinyl]acetic acid
(13c) (200 mg, 0.65 mmol) and ( )-4,5,6,7-tetrahydro-2H-
indazol-5-ylmethanamine dihydrochloride (1×2HCl) (146 mg,
0.65 mmol). The crude product was purified by column chro-
matography (silica gel, CH2Cl2/MeOH = 9:1) to give 80 mg
(28%) of white powder; mp 103-105 °C. IR (KBr): m 3331,
2924, 1652, 1581, 1479, 1443, 1434, 1226, 957, 748 cm–1.
MS (FAB): 442 (MH+, 91%), 149 (100%). 1H-NMR
(300 MHz, CDCl3): d = 0.85-0.96 (m, 1H, CH-5), 1.46-1.58
(m, 2H, CH2-6), 2.15-2.26, 2.57-2.68, 2.71-2.80 (3×m, 4H,
CH2-4, CH2-7), 3.12 (t, 2H, J = 6.78 Hz, Py-CH2), 3.25-3.38
(m, 2H, CH2NHCO), 3.85 (dt, 2H, J1 = J2 = 6.40 Hz,
CH2CH2NH), 4.83 (s, 2H, CH2CO), 6.15 (t br, 1H, PzNH),
6.68 (t br, 1H, NHCO), 7.01 (s, 1H, Pz-H), 7.16-7.21 (m, 2H,
2×Py-H), 7.30 (s, 1H, CH-3), 7.62 (m, Py-H), 8.58 (d, 1H,
J = 1.88 Hz, Py-H). Anal. C21H24N7O2Cl (C, H, N).
6.1.2.21. (-) 2-[6-Chloro-2-oxo-3-{[2-(2-pyridinyl)ethyl]-
amino}-1(2H)-pyrazinyl]-N-(4,5,6,7-tetrahydro-2H-indazol-
5-ylmethyl)acetamide (25). Using the general procedure
described above, 25 was prepared from 2-[6-chloro-2-oxo-3-
{[2-(2-pyridinyl)ethyl]amino}-1(2H)-pyrazinyl]acetic acid
(13c) (93 mg, 0.30 mmol) and (-)-4,5,6,7-tetrahydro-2H-
indazol-5-ylmethanamine (2) (45 mg, 0.30 mmol). The crude
product was purified by column chromatography (silica gel,
CH2Cl2/MeOH = 9:1) to give 50 mg (37%) of white powder;
6.1.2.19. ( ) 2-[6-Chloro-2-oxo-3-(phenethylamino)-1(2H)-
pyrazinyl]-N-(4,5,6,7-tetrahydro-2H-indazol-5-ylmethyl)ace-
tamide (21). General procedure for the synthesis of com-
pounds 21, 24 and 25 by coupling P3-P2 fragments 13b and
13c with ( )-4,5,6,7-tetrahydro-2H-indazol-5-ylmethanamine
dihydrochloride (1×2HCl) and (-)-4,5,6,7-tetrahydro-2H-
indazol-5-ylmethanamine (2). 2-[6-Chloro-2-oxo-3-(phen-
ethylamino)-1(2H)-pyrazinyl]acetic acid (13b) (200 mg,
0.65 mmol) and ( )-4,5,6,7-tetrahydro-2H-indazol-5-ylmetha-
namine dihydrochloride (1×2HCl) (146 mg, 0.65 mmol) were
dissolved in N,N-dimethylformamide (1.0 ml). 1-hydroxy-
benzo-triazole (90 mg, 0.67 mmol) and, after adjusting the
pH of the resulting solution to 8 with N-methylmorpholine,
1-(3-dimethylaminopropyl)-3’-ethyl-carbodiimide hydrochlo-
ride (125 mg, 0.65 mmol) were added. The reaction mixture
was stirred overnight at room temperature and partitioned
between ethyl acetate and saturated aqueous NaHCO3. The
organic layer was washed with brine, dried over Na2SO4, fil-
tered and the solvent removed in vacuo. The crude product
was purified by column chromatography (silica gel,
CH2Cl2/MeOH = 20:1) to give 60 mg (21%) of white pow-
der; mp 120-123 °C. IR (KBr): m 3344, 2924, 1655, 1648,
1583, 1487, 1240, 780, 694 cm–1. MS (FAB): 441 (MH+,
100%). 1H-NMR (300 MHz, CDCl3): d = 0.83-0.98 (m, 1H,
CH-5), 1.47-1.59 (m, 2H, CH2-6), 2.64-2.69, 2.71-2.80, 2.83-
2.88 (3×m, 4H, CH2-4, CH2-7), 2.96 (t, 2H, J = 7.16 Hz,
Ph-CH2), 3.27-3.40 (m, 2H, CH2NHCO), 3.68 (m, 2H,
CH2CH2NH), 4.83 (s, 2H, CH2CO), 6.00 (t br, 1H, Pz-NH),
6.11 (t br, 1H, NHCO), 7.03 (s, 1H, Pz-H), 7.22-7.37 (m, 6H,
Ph-H, CH-3) Anal. C22H25N6O2Cl × 0.5 H2O (C, H, N).
mp 101-104 °C;
␣
20 = -11.54° (c = 0.13, MeOH). The
͓ ͔
product was in all otheDr respects (IR, MS, 1H-NMR) identi-
cal with 24. Anal. C21H24N7O2Cl×H2O (C, H, N).
References
[1] J.I. Weitz, M. Crowther, Thromb. Res. 106 (2002) V275–V284.
[2] A.V. Khrenov, N.M. Ananyeva, J.H. Griffin, E.L. Saenko, Trends
Cardiovasc. Med. 12 (2002) 317–324.
[3] E.M. Antman, Eur. Heart J. Suppl. 6 (2004) B2–B8.
[4] T. Steinmetzer, J. Hauptman, J. Stürzebecher, Expert Opin, Invest.
Drugs 10 (2001) 845–864.
[5] C.A. Coburn, Exp. Opin. Ther. Patents 11 (2001) 721-738.
[6] J.B.M. Rewinkel, A.E.P. Adang, Curr. Pharm. Des. 5 (1999) 1043–
1075.
[7] K. Menear, Curr. Med. Chem. 5 (1998) 457–468.
[8] J. Hirsh, Thromb. Res. 109 (2003) S1–S8.
[9] C.S. Burgey, K.A. Robinson, T.A. Lyle, P.E.J. Sanderson, S.D. Lewis,
B.J. Lucas, J.A. Krueger, R. Singh, C. Miller-Stein, R.B. White,
B. Wong, E.A. Lyle, P.D. Williams, C.A. Coburn, B.D. Dorsey,
J.C. Barrow, M.T. Stranieri, M.A. Holahan, G.R. Sitko, J.J. Cook,
D.R. McMasters, C.M. McDonough, W.M. Sanders, A.A. Wallace,
F.C. Clayton, D. Bohn, Y.M. Leonard, T.J. Detwiler, J.J. Lynch,
Y.W. Yan, Z.G. Chen, L. Kuo, S.J. Gardell, J.A. Shafer, J.P. Vacca, J.
Med. Chem. 46 (2003) 461–473.
[10] A.E.P. Adang, A.P.A. de Man, G.M.T. Vogel, P.D.J. Grootenhuis,
M.J. Smit, C.A.M. Peters, et al., J. Med. Chem. 45 (2002) 4419–4432.
[11] J.M. Altenburger, G.Y. Lassalle, M. Matrougui, D. Galtier, J.C. Jetha,
Z. Bocskei, C.N. Berry, C. Lunven, J. Lorrain, J.P. Herault, P. Schaef-
fer, S.E. O’Connor, J.M. Herbert, Bioorg. Med. Chem. 12 (2004)
1713–1730.
6.1.2.20. ( ) 2-[6-Chloro-2-oxo-3-{[2-(2-pyridinyl)ethyl]-
amino}-1(2H)-pyrazinyl]-N-(4,5,6,7-tetrahydro-2H-indazol-
5-ylmethyl)acetamide (24). Using the general procedure
described above, 24 was prepared from 2-[6-chloro-2-oxo-3-
[12] D. Euler, P. Frech, S. Karki, C. Cowden, G. Pearce, P. Mehta, C. Lin-
demann, P. Byway, M. Wang, T. Gibson, Y. Cheng, G. Kwei, J. Rose,
Int. J. Pharm. 275 (2004) 19–27.

A. Kranjc et al. / European Journal of Medicinal Chemistry 40 (2005) 782–791
791
[13] S.B. Olsson and Executive Steering Committee on behalf of the
SPORTIF III Investigators, Lancet 362 (2003) 1691-1698.
[14] M.A. Crowther, J.I. Weitz, Expert, Opin. Inv. Drugs 13 (2004) 403–
413.
[15] K.E. Rittle, J.C. Barrow, K.J. Cutrona, K.L. Glass, J.A. Krueger,
L.C. Kuo, S.D. Lewis, B.J. Lucas, D.R. McMasters, M.M. Morris-
sette, P.G. Nantermet, C.L. Newton, W.M. Sanders,Y.Yan, J.P. Vacca,
H.G. Selnick, Bioorg. Med. Chem. Lett. 13 (2003) 3477–3482.
[16] B. Tomczuk, T. Lu, R.M. Soll, C. Fedde, A. Wang, L. Murphy,
C. Crysler, M. Dasgupta, S. Eisennagel, J. Spurlino, R. Bone, Bioorg.
Med. Chem. Lett. 13 (2003) 1495–1498.
[17] J.E. Reiner, D.V. Siev, G.-L. Araldi, J.J. Cui, J.Z. Ho, K.M. Reddy,
L. Mamedova, P.H. Vu, K.S.S. Lee, N.K. Minami, T.S. Gibson,
S.M. Anderson, A.E. Bradbury, T.G. Nolan, J.E. Semple, Bioorg.
Med. Chem. Lett. 12 (2002) 1203–1208.
[18] J.J. Cui, G.-L. Araldi, J.E. Reiner, K.M. Reddy, S.J. Kemp, J.Z. Ho,
D.V. Siev, L. Mamedova, T.S. Gibson, J.A. Gaudette, N.K. Minami,
S.M. Anderson, A.E. Bradbury, T.G. Nolan, J.E. Semple, Bioorg.
Med. Chem. Lett. 12 (2002) 2925–2930.
[29] L. Peterlin-Mašicˇ, D. Kikelj, A. Jurca, P. Marinko, A. Trampuš-
Bakija, M. Stegnar, D. Ðelovicˇ, WO Patent Application 03/048155,
2003; Chem. Abstr. 2003, 139, 36537.
[30] R.C.A. Isaacs, K.J. Cutrona, C.L. Newton, P.E.J. Sanderson,
M.G. Solinsky, E.P. Baskin, I.W. Chen, C.M. Cooper, J.J. Cook,
S.J. Gardell, S.D. Lewis, R.J. Lucas, E.A. Lyle, J.J. Lynch, A.M. Nay-
lor-Olsen, M.T. Stranieri, K. Vastag, J.P. Vacca, Bioorg. Med. Chem.
Lett. 8 (1998) 1719–1724.
[31] P.E.J. Sanderson, K.J. Cutrona, B.D. Dorsey, D.L. Dyer,
C.M. McDonough, A.M. Naylor-Olsen, I.W. Chen, Z.G. Chen,
J.J. Cook, S.J. Gardell, J.A. Krueger, S.D. Lewis, J.H. Lin, B.J. Lucas,
E.A. Lyle, J.J. Lynch, M.T. Stranieri, K. Vastag, J.A. Shafer,
J.P. Vacca, Bioorg. Med. Chem. Lett. 8 (1998) 817–822.
[32] P.E.J. Sanderson, T.A. Lyle, K.J. Cutrona, D.L. Dyer, B.D. Dorsey,
C.M. McDonough, A.M. Naylor-Olsen, I.W. Chen, Z.G. Chen,
J.J. Cook, C.M. Cooper, S.J. Gardell, T.R. Hare, J.A. Krueger,
S.D. Lewis, J.H. Lin, B.J. Lucas, E.A. Lyle, J.J. Lynch, M.T. Stranieri,
K. Vastag,Y.W.Yan, J.A. Shafer, J.P. Vacca, J. Med. Chem. 41 (1998)
4466–4474.
[19] L. Peterlin-Mašicˇ, D. Kikelj, Tetrahedron Lett. 41 (2000) 5589–5592.
[20] P. Marinko, A. Obreza, L. Peterlin, A. Krbavcˇicˇ, D. Kikelj, J. Hetero-
cycl. Chem. 37 (2000) 405–409.
[21] P. Marinko, J. Kastelic, A. Krbavcˇicˇ, D. Kikelj, Tetrahedron Lett. 42
(2001) 8911–8913.
[33] J.J. Parlow, R.G. Kurumbail, R.A. Stegeman, A.M. Stevens,
W.C. Stallings, M.S. South, J. Med. Chem. 46 (2003) 4696–4701.
[34] J.J. Parlow, B.L. Case, T.A. Dice, R.L. Fenton, M.J. Hayes,
D.E. Jones, W.L. Neumann, R.S. Wood, R.M. Lachance, T.J. Girard,
N.S. Nicholson, M. Clare, R.A. Stegeman, A.M. Stevens, W.C. Stall-
ings, R.G. Kurumbail, M.S. South, J. Med. Chem. 46 (2003) 4050–
4062.
[22] L. Peterlin-Mašicˇ, A. Jurca, P. Marinko, A. Jancˇar, D. Kikelj, Tetrahe-
dron 58 (2002) 1557–1563.
[23] L. Peterlin-Mašicˇ, D. Kikelj, Tetrahedron 57 (2001) 7073–7105.
[24] L. Peterlin-Mašicˇ, G. Mlinšek, T. Šolmajer, A. Trampuš-Bakija,
M. Stegnar, D. Kikelj, Bioorg. Med. Chem. Lett. 13 (2003) 789–794.
[25] P. Marinko,A. Krbavcˇicˇ, G. Mlinšek, T. Šolmajer,A. Trampuš-Bakija,
M. Stegnar, J. Stojan, D. Kikelj, Eur. J. Med. Chem. 39 (2004)
257–265.
[35] C.S. Schneider, J. Mierau, J. Med. Chem. 30 (1987) 494–498.
[36] P.E. Sanderson, A.M. Naylor-Olsen, D. Dyer, J.P. Vacca,
R.C.A. Isaacs, B.D. Dorsey, M.E. Fraley, WO 97/01338, 1997, Chem.
Abstr. 126 (1997) 171490.
[37] K. Hilpert, J. Ackermann, D.W. Banner, A. Gast, K. Gubernator,
P. Hadvary, J. Med. Chem. 37 (1994) 3889–3901.
[26] D. Kikelj, L. Peterlin-Mašicˇ, P. Marinko, M. Breznik, M. Stegnar, A.
Trampuš-Bakija, M. Fortuna, WO PatentApplication 01/85760, 2001;
Chem. Abstr. 2001, 135, 358167.
[38] Y. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099–3108.
[39] R.B. Brandt, J.E. Laux, S.W. Yates, Biochem. Med. Metab. Biol. 37
(1987) 344–349.
[27] L. Peterlin-Mašicˇ, A. Kranjc, P. Marinko, G. Mlinšek, T. Šolmajer,
M. Stegnar, D. Kikelj, Bioorg. Med. Chem. Lett. 13 (2003) 3171–
3176.
[28] A. Kranjc, L. Peterlin-Mašicˇ, J. Ilaš, A. Preželj, M. Stegnar, D. Kikelj,
Bioorg. Med. Chem. Lett. 14 (2004) 3251–3256.
[40] D. Gustafsson, T. Antonsson, R. Bylund, U. Eriksson, E. Gyzander,
I. Nilsson, M. Elg, C. Mattson, J. Deinum, S. Pehrsson, O. Karlsson,
A. Nisson, H. Sörensen, Thromb. Haemost. 79 (1998) 110–118.
[41] M.A. Crowther, J.I. Weitz, Expert Opin, Inv. Drugs 13 (2004)
403–413.